GLYCOSYLATED AND NON-GLYCOSYLATED BACTERICIDAL/PERMEABILITY INCREASING PROTEINS, AND METHODS FOR PRODUCING SAME

Abstract: The subject invention provides a method for producing non-glycosylated Bactericidal/Permeability Increasing Protein using a gram-negative bacterial cell, which comprises transfecting the cell with a vector encoding Bactericidal/Permeability Increasing Protein; culturing the cell so transfected under conditions permitting the production of non-glycosylated Bactericidal/Permeability Increasing Protein in the cell; and recovering from the cell the non-glycosylated, Bactericidal/Permeability Increasing Protein so produced. Finally, the subject invention provides the non-glycosylated Bactericidal/Permeability Increasing Protein produced by the method of the subject invention.

Description: GLYCOSYIATED AND NON-GLYCOSYLATED BACTERICIDAL/PERMEABILITY INCREASING PROTEINS. AND METHODS FOR PRODUCING SAME This application is a continuation-in-part of U.S. Serial No. 07/567,016, filed August 13, 1990, which is a continuation-in- part of U.S. Serial No. 07/468,696, filed January 22, 1990, now U.S. Patent No. 5,089,274, issued February 18, 1992, which is a continuation-in-part of U.S. Serial No. 07/310,842, filed February 14, 1989, now abandoned, the contents of which are hereby incorporated by reference. Background of the Invention Gram-negative infections are a major cause of morbidity and mortality, especially in hospitalized and immunocompromised patients. [Duma, R.J., Am. J. of Med. , 78 (Suppl. 6A) : 154-164 (1985); and Kreger B.E., D.E. Craven and W.R. McCabe, Am. J. Med., 68: 344-355 (1980)]. Although available antibiotics are effective in containing gram-negative infections, they do nothing to neutralize the pathophysiological effects associated with lipopolysaccharide (LPS) . LPS, also referred to herein as endotoxin, is a major component of the outer membrane of gram-negative bacteria and is released when the organisms are lysed. [Ahenep, J.L. and K.A. Morgan/ J. Infect. Dis., 150 (3): 380-388 (1984)]. LPS released during antibiotic therapy is a potent stimulator of the inflammatory response. Many detrimental effects of LPS in vivo result from soluble mediators released by inflammatory cells. [Morrison D.C. and R.J. Ulevich, Am. J. Pathol., 93 (2) : 527-617 (1978)] . LPS induces the release of mediators by host .inflammatory cells which may ultimately result in disseminated intravascular coagulation (DIC) , adult respiratory distress syndrome (ARDS) , renal failure ' and irreversible shock. Monocytes and neutrophilic granulocytes play a key role in host defense against bacterial infections and also participate in the pathology of endotoxemia. These cells ingest and kill microorganisms intracellularly and also respond to LPS in vivo and in vitro by releasing soluble proteins with microbicidal, proteolytic, opsonic, pyrogenic, complement-activating and tissue-damaging effects. Tumor necrosis factor (TNF) , a cytokine released by LPS-stimulated monocytes, mimics some of the toxic effects of LPS in vivo. Injecting animals with TNF causes fever, shock and alterations in glucose metabolism. TNF is also a potent stimulator of neutrophils. Other cytokines, such as IL-1, IL-6 and IL-8, are also released and may play a role in the inflammatory response to LPS. Soluble LPS causes decreased neutrophil chemotaxis, increased adhesiveness, elevated hexose monophosphate shunt activity and 0 2 radical production, upregulation of surface receptors for complement, and release of granule proteins into the surrounding medium. [Morrison and Ulevich (1978)]. Both specific and azurophil compartments degranulate in response to LPS. [Bannatyne, R.M. , N.M. Harnett, K.Y. Lee and W.D. Rigger, J. Infect. Dis., 156 (4) :469-474 (1977)]. Azurophil proteins released in response to LPS may be both harmful and beneficial to the host. Neutrophil elastase causes degradation of protease inhibitors responsible for suppressing the coagulation cascade. This results in coagulopathies such as disseminated intravascular coagulation, a potentially lethal consequence of endotoxemia. Azurophil granules also contain bactericidal molecules such as myeloperoxidase and Bactericidal/Permeability Increasing Factor, hereinafter referred to as BPI Protein. Rabbit .BPI was first discovered in 1975. [Weiss, J., R.C. Franson, S. Becherdite, K. Schmeidler, and P. Elsbach, J. Clin. Invest., 55:33 (1975)]. BPI Protein was isolated from human neutrophils in 1978. [Weiss, J. , P. Elsbach, I. Olson and H. Odeberg, J. Biol. Chem, 253 (8) :2664-2672 (1978)]. In 1984 a 57 kD protein with similar properties was isolated from human neutrophils. [Shafer, W.M. , C.E. Martin and J.K. Spitznagel, Infect. Immun., 45:29 (1984)]. This protein is identical to BPI Protein as determined by the 57 kDa protein's N-Terminal sequence, amino acid composition, molecular weight and source. Although, the authors were unable to reproduce the chromatographic isolation procedure used by Elsbach, et al. and Weiss, et al. Human BPI Protein is a 57 kD protein which binds to the outer membrane of susceptible gram-negative bacteria. [Weiss, et al. (1978)]. The fact that BPI Protein is a Lipid A-binding protein is evidenced by: (1) rough strains of bacteria are more sensitive to both bactericidal and permeability increasing activities of BPI Protein [Weiss, J., M. Hutzler and L. Kao, Infect. Immun., 51:594 (1986)]; (2) mutations in Lipid A cause decreased binding and increase resistance to bactericidal activity of both polymyxin B and BPI Protein [Farley, M.M. , W.M. Shafer and J.K. Spitznagel, Infect. Immun., 56:1589 (1988)]; (3) BPI Protein competition with polymyxin B (PMB) for binding to S. typhimurium [Farley 1988] ; and (4) BPI Protein sequence homology and immunocross- reactivity to another LPS-binding protein Lipopolysaccharide Binding Protein (LBP) . LBP-LPS complexes have been shown to stimulate the oxidative burst on neutrophils in response to formulated peptides. In addition, LBP-LPS complexes bind back to a cell surface receptor on monocytes (CD 14) resulting in the induction of tumor necrosis factor (TNF) production. Thus, LBP mediates the immunostimulatory activity of LPS and therefore stimulates the toxic response to endotoxin. High density lipoprotein (HDL) , another LPS binding protein found in human serum in complex with LPS, does not show the stimulatory effect on neutrophils. BPI Protein binding to gram-negative bacteria disrupts LPS structure, alters microbial permeability to small hydrophobic molecules and causes cell death. [Weiss, et al., (1978)]. BPI Protein kills bacteria under physiologic conditions of pH and ionic strength in vitro, indicating that it may be active in vivo outside the low pH environment of the phagolysosome. All of the bactericidal and permeability increasing activities of BPI Protein are present in the N-terminal 25kD fragment of the protein. [Ooi, C.E., J. Weiss, P. Elsbach, B. Frangione, and B. Marrion, J. Biol. Chem., 262: 14891 (1987)]. It was previously understood that the beneficial effects of BPI Protein are limited to its bactericidal effects. However, it was later shown that BPI Protein also binds to endotoxin but that, unlike LBP, BPI Protein inhibits the immunostimulatory activities of LPS rather than promoting them. Mannion, et al. [Preferential Binding of the Neutrophil Cytoplasmic Granule-Derived Bactericidal/Permeability Increasing Protein to Target Bacteria, J. Immunol. 142:2807 (1989)] showed that BPI Protein could be purified from neutrophil granule extracts by binding to gram-negative bacteria. Eighty percent of the bound BPI Protein could then be eluted with 200 mM MgCl 2 , in 10 mM sodium acetate pH 4.0 at 37°C and the bacteria removed by centrifugation. Expression of many proteins in bacteria, such as E. coli. has proved to be difficult. Which proteins will be expressed in E. coli and whether or not the protein will be expressed in the form of a denatured insoluble inclusion body or folded into active form cannot be determined ahead of time. A variety of conditions can be explored to shift the amount of insoluble vs. soluble material, including fermentation conditions such as media composition, pH, ionic strength, and temperature [A. Mitraki and J. King Bio/Technology 7:690-697, 1989, or CH. Schein Bio/Technology 7:1141-1149 (1989)]. Further, for proteins in insoluble inclusion body form, a variety of techniques can be explored to recover renatured active protein (BioTechniques 3:298-306 (1985)), but with success very much dependent on the character of the protein in question. The subject invention provides, inter alia, a method for producing Bactericidal/Permeability Increasing Protein, which includes BPI Protein, using gram-negative bacteria. BPI Protein is a protein possessing antibiotic functions, i.e., potent antibacterial activity against gram-negative bacteria. Accordingly, one skilled in the art would not expect to be able to express a protein in active soluble form in a host cell, which protein functions as a known cytotoxin against the host cell. Indeed, the fact that BPI Protein kills gram- negative bacteria has prevented others in the field from developing a bacterial expression system for BPI Protein. The method of the subject invention represents a substantial advance over the state of the art, since the production of recombinant proteins in gram-negative bacteria, such as E. coli. can be up to 100 times more cost effective than other production methods such as mammalian cell expression systems. Suπimarv of the Invention The subject invention provides a method for producing non- glycosylated Bactericidal/Permeability Increasing Protein using a gram-negative bacterial cell, which comprises transfecting the cell with a vector encoding Bactericidal/Permeability Increasing Protein; culturing the cell so transfected under conditions permitting the production of non-glycosylated Bactericidal/Permeability Increasing Protein in the cell; and recovering from the cell the non- glycosylated, Bactericidal/Permeability Increasing Protein so produced. In the preferred embodiment, the Bactericidal/Permeability Increasing Protein has no leader peptide attached thereto when expressed. In the preferred embodiment, the conditions permitting the production of non-glycosylated Bactericidal/Permeability Increasing Protein in the cell comprise low temperature. In one embodiment, the recovering comprises the steps of (a) lysing the cell under conditions preventing the formation of a complex between the Bactericidal/Permeability Increasing Protein and LPS so as to release the Bactericidal/ Permeability Increasing Protein from the cell; and (b) isolating the Bactericidal/Permeability Increasing Protein so released. In another embodiment, the recovering comprises the steps of (a) lysing the cell under conditions permitting the formation of a complex between the Bactericidal/Permeability Increasing Protein and LPS; (b) isolating the resulting Bactericidal/ Permeability Increasing Protein-LPS complex; (c) dissociating the Bactericidal/Permeability Increasing Protein- PS complex so isolated, so as to release the Bactericidal/Permeability Increasing Protein from the Bactericidal/Permeability Increasing Protein-LPS complex; and (d) isolating the Bactericidal/Permeability Increasing Protein so released. Finally, the subject invention provides the non-glycosylated Bactericidal/Permeability Increasing Protein produced by the method of the subject invention. Brief Description of the Figures Figure 1 Schematic drawing of cDNA encoding BPI Protein. Figure 2 A nucleotide and amino acid sequence of BPI Protein mutagenic primer 25 kDa Pro 212 TGA which is a C-terminal truncation of BPI Protein. Figure 3 A nucleotide and amino acid sequence of BPI Protein mutagenic primer 38 kDa Pro 337 TGA which is a C-terminal truncation of BPI Protein. Figure 4 A nucleotide and amino acid sequence of BPI Protein mutagenic primer: preferred ATG 5' Hindlll which is a C-terminal truncation of BPI Protein. Figure 5 A schematic drawing of pSVBPIMDH. Figures 6-1 and 6-2 A schematic drawing of pAc373. Figure 7 SDS-PAGE analysis of (l) nBPI Protein (#148104), (2) rBPI Protein (#148159), and (3) rBPI Protein (#148179). Figures 8A-1 and 8A-2 Amino acid sequence of BPI Protein. Figures 8B-1 and 8B-2 cDNA sequence of BPI Protein. Figure 9 Protein sequence for p337. Figure 10 Protein sequence for p212. Figure 11 SDS PAGE analysis of BPI Protein produced in mammalian cells and in E. coli. and of BPI Protein obtained through non- recombinant methods. Lanes 1 and 10 are molecular weight markers; lane 3 is BPI Protein produced in CHO cells; lane 5 is naturally derived BPI Protein; lane 7 is BPI Protein produced in E. coli and partially purified. Figure 12 Assay comparing TNF release in whole blood for BPI Protein produced in CHO cells and E. coli. For the TNF assay, blood is drawn with citrate anticoagulant from healthy donors. BPI Protein is added, then LPS is added. Incubation is carried out for four hours at 37°C with 5% C0 2 . Samples are centrifuged and plasma collected. TNF is assayed using a sandwich assay. Figure 13 Activity in LAL assay of BPI Protein produced in E. coli. Detailed Description of the lnv-.τ._--_lon Specifically, the subject invention provides a method for producing non-glycosylated Bactericidal/Permeability Increasing Protein using a gram-negative bacterial cell, which comprises transfecting the cell with a vector encoding Bactericidal/ Permeability Increasing Protein; culturing the cell so transfected under conditions permitting the production of non-glycosylated Bactericidal/Permeability Increasing Protein in the cell; and recovering from the cell the non- glycosylated, Bactericidal/Permeability Increasing Protein so produced. As used herein, Bactericidal/Permeability Increasing Protein means BPI Protein and BPI variants. As used herein, BPI Protein means the native 55 kD human protein having the amino acid sequence shown for human BPI Protein in Figures 8A-1 and 8A-2, and encoded by the cDNA sequence shown in Figures 8B-1 and 8B-2. As used herein, a BPI variant means a protein comprising a portion of BPI Protein, which protein is capable of (a) binding to LPS, (b) competing with BPI Protein or LBP for binding to LPS, and (c) inhibiting the LPS-mediated production of TNFα by human monocytes. BPI variants include, but are in no way limited to, a fragment of BPI Protein, a point mutant of BPI Protein, a deletion mutant of BPI Protein, or a point and deletion mutant of BPI Protein. Specific BPI variants include, but are in no way limited to, B (S351 . >A) (glycosylation site deleted) ; B^ OODD (acid cleavage site inserted) ; B 99 (N-terminal domain of BPI Protein) ; B 2(XM56 (C- terminal fragment of BPI Protein) ; B (F61 - >Q (cysteine insertion) ; B (CI32 _ >A. (cysteine deletion) ; B (C132 _ >S) (cysteine deletion) ; B (C135 . >S) (cysteine deletion) ; B (C . 17J . >S) (cysteine deletion) ; B (C i 32 _ >A)(C135 . > s )( ci 75 - > s ) (multiple cysteine deletions) ; B (C . 132 . >S)(C135 . >S)(C175 . >S) (multiple cysteine deletions) . In the BPI variant designations supra. the portion of BPI Protein in the variants is designated by the letter B, followed by an amino acid sequence numbering assignment corresponding to that shown in Figures 8A-1, 8A-2, 8B-1 and 8B-2 for human BPI Protein, wherein the mature N-terminus is designated as residue 1. . Single amino acid residue substitutions are noted in parentheses, wherein the original amino acid residue is indicated (using the standard one letter code for amino acids) , followed by the substitute amino acid residue. For example, the BPI variant wherein the original serine residue at position 351 is substituted with an alanine residue (which substitution removes a glycosylation signal) is designated B (S351 - >A) . The amino acid substitutions may be a substitution wherein an original amino acid residue at a given position is substituted with the residue at the corresponding position in a different protein. CKn . >Y) is an example of such a substitution, wherein amino acid residue X at position n in BPI Protein is substituted with residue Y which is found at position n in LBP (or rabbit or bovine LBP) . Amino acid residue insertion changes are noted in parentheses, by indicating the amino acid residue after which the insertion occurs, followed by the amino acid residue after which the insertion occurs together with the inserted residue or residues. For example, in B (Dm . >OP) , a proline residue is inserted after the aspartate residue at position 199. BPI variants having one or more cysteine residues deleted or substituted with serine or another amino acid residues would be useful for preventing the aggregation of BPI variants during their production or use. The BPI variants may have one or more non-conserved glycosylation sites deleted. Alternatively, the BPI variants may have one or more non-conserved glycosylation sites inserted. The BPI variants may have one or more secondary structure- altering amino acid residues deleted or added. For example, one or more of the non-conserved proline residues in BPI Protein may be substituted with a non-proline residue. As used herein, LPS means lipopolysaccharide. As used herein, TNFo; means tumor necrosis factor alpha. In one embodiment, the gram-negative bacterial cell is E ^ _ coli. Methods of transfecting gram-negative cells, vectors suitable for transfecting gram-negative cells, and methods of culturing gram-negative cells are all well known to those skilled in the art. Examples of such methods and vectors are provided infra. In the preferred embodiment, the Bactericidal/Permeability Increasing Protein has no leader peptide attached thereto when expressed. As used herein, the term "when expressed" means when first translated from the mRNA. In the preferred embodiment, the conditions permitting the production of non-glycosylated Bactericidal/Permeability Increasing Protein in the cell comprise low temperature. As used herein, the term "low temperature" means a temperature between 25-36°C. In the preferred embodiment, low temperature means a temperature of about 26°C. The Bactericidal/Permeability Increasing Protein may be produced in active soluble form. Two embodiments are provided infra for recovering Bactericidal/Permeability Increasing Protein produced in active soluble form. In one embodiment, the recovering comprises the steps of (a) lysing the cell under conditions preventing the formation of a complex between the Bactericidal/Permeability Increasing Protein and LPS so as to release the Bactericidal/Permeability Increasing Protein from the cell; and (b) isolating the Bactericidal/Permeability Increasing Protein so released. In another embodiment, the recovering comprises the steps of (a) lysing the cell under conditions permitting the formation of a complex between the Bactericidal/Permeability Increasing Protein and LPS; (b) isolating the resulting Bactericidal/ Permeability Increasing Protein-LPS complex; (c) dissociating the Bactericidal/Permeability Increasing Protein-LPS complex so isolated, so as to release the Bactericidal/Permeability Increasing Protein from the Bactericidal/Permeability Increasing Protein-LPS complex; and (d) isolating the Bactericidal/Permeability Increasing Protein so released. In one embodiment lysing the cell comprises removing the outer membrane of the cell, and subsequently breaking open the resulting cell. In one embodiment, dissociating the Bactericidal/Permeability Increasing Protein-LPS complex comprises contacting the complex with divalent cations. Contacting the complex with divalent cations may comprise contacting the complex with 0.05M - 1.0M MgCl 2 . In the preferred embodiment, contacting the complex with divalent cations comprises contacting the complex with 0.5M MgCl 2 . Alternatively, the Bactericidal/Permeability Increasing Protein may be produced in inactive, insoluble form and be stored in inclusion bodies. The embodiment provided infra is for recovering Bactericidal/Permeability Increasing Protein produced in inactive, insoluble form. In this embodiment, the recovering comprises the steps of (a) extracting from the inclusion bodies the Bactericidal/ Permeability Increasing Protein in its inactive, insoluble form, and (b) refolding the inactive, insoluble protein so as to generate active, soluble Bactericidal/Permeability Increasing Protein. Examples of this embodiment are provided infra. The subject invention still further provides a method for producing non-glycosylated Bactericidal/Permeability Increasing Protein using a prokaryotic cell, which comprises transfecting the cell with a vector encoding Bactericidal/ Permeability Increasing Protein; culturing the cell so transfected under conditions permitting the production of non- glycosylated Bactericidal/Permeability Increasing Protein in the cell; and recovering from the cell the non-glycosylated, Bactericidal/Permeability Increasing Protein so produced. In one embodiment, the prokaryotic cell is a gram-positive cell. Finally, the subject invention provides the non-glycosylated Bactericidal/Permeability Increasing Protein produced by the method of the subject invention. The subject invention is illustrated in the Experimental Details section which follows. This section is set forth to aid in understanding the subject invention, but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow. Experimental Details Example 1 Purification and characterization of Neutrophil BPI Protein A. Materials and Methods 1. Reagents Lipopolysaccharide from E. coli 0111:B4, S. typhimurium wild type, glycolipid from S. typhimurium RE mutant, and Lipid A from S. typhimurium RE mutant, and LPS from P. aeruginosa were purchased from RIBI Immmunochem Research, Inc. (Hamilton, MT) ; Fmet-Leu-Phe (FMLP) and polymyxin B Sulfate were purchased from Sigma Chemical Co. (St. Louis, MO); Hank's Balanced Salt Solution without calcium, magnesium and phenol red (HBSS) were purchased from Hazelton Research Products (Denver, PA) ; Ficoll-Paque, Percoll and Macrodex were purchased from Pharmacia, Inc. (Piscataway, NJ) ; TNF and anti-TNF were purchased from Endogen (Boston, MA) ; Fluorescein-conjugated goat anti-mouse IgG was purchased from TAGO, Inc. (Burlingame, CA) ; IgGl control antibody was purchased from Coulter Immunology (Hialeah, FL) ; Phycoerythrin (PE) conjugated anti- CR3 (Leu-15) and IgG2a control were purchased from Becton Dickinson (Mountain View, CA) ; and anti-CRl monoclonal antibodies may be obtained commercially or according to methods known to those skilled in the art. 2. Azurophil Granule Isolation and Extraction Granulocytes were isolated from buffy coats obtained from local blood banks. Buffy coats were diluted 3-4X in HBSS and granulocytes were separated from mononuclear cells by centrifugation through 64% Percoll. The pellet was subjected to diisopropylfluorophosphate (DFP) , washed, and resuspended in ice cold lysis buffer (10 mM PIPES, pH 6.8, 100 mM C1, 3mM NaCl, 3.5 mM MgC12) and disrupted by nitrogen cavitation (Parr Instrument Co., Moline, IL) . Azurophil granules were isolated on discontinuous Percoll gradients as described by Borregaard. [Borregaard, N. , J.M. Heiple, E.R. Simons, and R.A. Clark, J. Cell. Biol., 97: 52-61 (1983)]. The azurophil granules were collected and Percoll was removed by centrifugation at 180,000 X g for 2 hours. The granules were lysed by 4 cycles of freeze-thaw followed by 1 minute of sonication. The lysed granules were extracted in an equal volume of 100 mM glycine, pH 2 by vortexing intermittently for 1 hour at room temperature. The acid extract was clarified by centrifugation at 30,000 X g for 20 minutes and at 200,000 X g for 30 minutes. 3. Neutrophil Isolation Venous blood was drawn from healthy volunteer donors into acid citrate dextrose anticoagulant and immediately placed on ice. Five parts blood were mixed with 1 part cold Macrodex, and allowed to settle for 1.5 - 2 hours at 4°C. Leukocyte-rich plasma was washed IX in cold HBSS, then resuspended in HBSS and layered over Ficoll-Paque. If significant erythrocyte contamination was present, the granulσcyte pellet was subjected to hypotonic lysis. The cells were washed 2X in HBSS and resuspended in HBSS + 2% autologous plasma to give a final granulocyte concentration of 1 X 10 6 /ml in the incubation mixture. 4. BPI Protein Purification Approximately 2 mg of crude azurophil granule extract was separated by size on a Biosil (TSK-250) (7.8 mm x 600 mm) high performance size exclusion column using 50 mM glycine and 100 mM NaCl buffer, pH 2.0, under isocratic conditions of a flow rate of 1 ml/min. Column fractions with the greatest LPS inhibitory activity contained a large proportion of the 54 kD species as shown by SDS PAGE. These TSK fractions were pooled and run over an Aquapore weak cation exchange (WCX) column (2.1 mm X 30 mm) using 50 mM citrate, pH 5.5, and eluted in a gradient of 0-75%, of 50 mM citrate and 1 M NaCl (Buffer B) in 25 min, then 75-100% Buffer B in 5 min with a flow rate of 200 ml/min. Material of 55 kD was recovered from cation exchange and appeared as a single band on SDS page. In some experiments BPI Protein was further purified by reverse phase HPLC on a Vydac C4 column loaded for 12 min in 0.1% CH 3 CN plus 0.1% TFA, in 30 min with a flow rate of 200 ml/min (Rainin Instruments, Emeryville, CA) . 5. Neutrophil stimulation Isolated neutrophils were kept on ice until incubated with and without stimuli at 37°C for 30 minutes. Following the incubation, cells were washed in a large volume of cold PBS + 0.05% Na Azide + 2% autologous plasma. Pellets were divided in two, one stained with 50 μl control IgGl antibody (20 μg/1 x 10 6 cells) , the other with 50 μl of 20 μg/1 x 10 6 cells anti-CRl for 30 minutes at 0°C. Following this incubation the cells were washed 2X with PBS + autologous plasma, then stained with goat-anti-mouse IgG-FITC, and in some experiments, 20 μl of IgG2a-phycoerythrin (PE) in control wells, and 20 μl Leu-15 PE in test wells. Following a 30 minute incubation at 0°C and 2 more washes, the cells were analyzed by flow cytometry on a Becton Dickinson FACStar flow cytometer (Becton Dickinson, Mountain View, CA) . Neutrophil stimulation was measured by comparing mean fluorescence intensity of samples which had been incubated in HBSS + 2% autologous plasma alone (control) to those incubated with LPS or LPS which had been preincubated for 30 minutes at 37°C with BPI Protein or polymyxin B. Data are expressed as % stimulation or % inhibition and were calculated using the mean fluorescence intensity (Fl) , on a log scale, according to: % Stimulation = [(Experimental - Control)/(Maximum - control)] X 100 and % Inhibition = 1-[(+Inhibitor)-(Control)]/[(-Inhibitor)-(Control)] X 100. 6. Amino Acid Analysis Vapor phase hydrolysis of BPI Protein and amino acid derivitization was performed using a Pico-tag Workstation (Waters, Milford MA) and chromatographic analysis of the phenylthiocarbamyl amino acids was performed on an applied Biosystems 130 A MPLC using Protocols provided by the manufacturer. 7. Sequence Analysis BPI Protein N-terminal sequence was analyzed by automated Edman degradation using an applied Biosystems 477A pulse liquid phase sequenator (Applied Biosystems, Foster City, CA) . Phenylthiohydantoin amino acid analysis was performed on line using an applied biosystems Model 120A liquid chromatograph. B. Results Human neutrophils may be stimulated both in vivo and in vitro by lipopolysaccharide. Upon activation, surface expression of receptors for C3b and C3bi (CR1 and CR3, respectively) increases. Using the Fluorescence Activated Cell Sorter (FACS) , fluorescence intensity of freshly isolated human neutrophils was measured following stimulation with increasing doses of 0111:B4 LPS (data not shown) . Because commonly observed maximum stimulation was at or above 10 ng/ml, experiments testing for inhibition of 0111:B4 LPS used 10 ng/ml as the stimulatory dose. All experiments were performed in duplicate. In most experiments, data is shown only for CR1 since no conditions were observed wherein neutrophil stimulation caused upregulation of CR1 or CR3 alone [M. Marra et al., J. Immunol. 144 (2) :662-666 (1990)]. To determine whether proteins found in neutrophil azurophil granules could interfere with the neutrophil response to LPS, crude acid extracts of azurophil granules were pre-incubated with LPS for 30 minutes at 37°C. The mixture was then tested for its ability to stimulate neutrophils. Azurophil protein (1 μg/ml) could effectively block stimulation of 1 X 10 6 polymorphonuclear leukocytes (PMN)/ml by 10 ng/ml of LPS (data not shown) . This effect was not observed using glycine extraction buffer preincubated with LPS, nor was there any stimulation of neutrophils using crude extract or glycine buffer control (data not shown) . To further investigate which of the proteins in the extract was/were responsible for inhibitory effect, crude acid extracts were separated by reverse phase HPLC, and each peak was assayed separately for LPS inhibitory activity. The identity of each of the peaks was previously determined using a two-dimensional purification approach involving microbore reverse phase HPLC in the first dimension followed by SDS PAGE, electroblotting and microsequencing. The azurophil proteins can be resolved into 10 discrete peaks whose identities are shown in Table 1. The amino acid sequences shown are for the first 15 amino acids of the N-terminal. LPS inhibitory activity of 1 μg of each peak was tested. Peak 9 had the highest LPS neutralizing activity. The major protein species in this peak has N-terminal identity with BPI Protein described previously [Weiss, J. , P. Elsbach, I. Olsson and H. Odeberg, J. Biol. Chem, 253 (8) :2664-2672 (1978)]. BPI Protein has been shown to contain the majority of the gram- negative bactericidal activity in azurophil granule protein extracts. Cathepsin G showed some inhibition of LPS, but the data between experiments were not as reproducible as for peak 9. Cathepsin G has been shown to bind to LPS in vitro and to kill gram-negative organisms, although to a lesser extent than BPI Protein. Other proteins which have demonstrated microbicidal activity against gram-negative organisms are elastase and the defensins. However, these proteins (1 μg/ml) did not inhibit the stimulatory activity of LPS on neutrophils. LPS inhibitory activity of crude azurophil extracts was further characterized and purified using size exclusion and ion exchange followed by reverse phase chromatography. LPS inhibitory activity comigrates with a pure 55 kD band seen on SDS PAGE (data not shown) . Because the buffer used in the RPLC separations (CH 3 CN and 0.1% trifluoroacetic acid (TFA) ) significantly diminishes the LPS inhibitory activity of BPI Protein (data not shown) , and since the material purified from ion exchange chromatography was of high purity as judged by SDS PAGE, size exclusion/ion exchange material was used to generate a dose response curve. Table 1 Azurophil Granule-Derived Proteins Peak Identity 1 5 10 15 I I I I 1 Defensins C Y C R I P A C I A G E R R Y (HNP-2) 2 Granulocidin V C S C R L V F C R R T G L R (HNP-4) 3 Eosinophil Cationic Protein (ECP) X P P Q F T R A Q W F A I Q H 4a Eosinophil - Derived Neurotoxin (EDN) K P P Q F T X A Q X F E T Q X 4b Cathepsin G I I G G R E S R P H S R P Y M 5a Lysozyme K V F E R X E L A R T L K R L 5b Eosinophil Major Protein (MBP) T C R Y L L V R S L Q T F S Q 6 Unknown I V G G R K A R P X Q F P F L 7 Unknown I V G G H E A Q P H S R P Y M 8a Myeloperoxidase V N C E T S C V Q Q P P C F P 8b Elastase I V G G R R A R P H A X P F M 9 BPI Protein V N P G V V V R I S Q K G L D This size exclusion/ion exchange purified material was confirmed to be BPI Protein by N-terminal sequence analysis. Protein concentration was determined by amino acid analysis. About 90 ng/ml of BPI Protein is required for maximal inhibition of the neutrophil response to 10 ng/ml 0111/B4 LPS. The neutrophil response to formulated peptide (10 "7 M FMLP) was not inhibited by BPI Protein (data not shown) . A similar dose response curve for the polypeptide antibiotic Polymyxin B (PMB) was seen. Polymyxin B binds to the Lipid A moiety of LPS and neutralizes some of its toxic effects both in vivo and in vitro. Polymyxin B binds to LPS stoichio- metrically [Morrison, D.C. and D.M. Jacobs, Immunochem, 13: 813-818 (1976)]. The calculated amount of PMB required to inhibit 10 ng/ml of smooth LPS is approximately 0.67 nM. In the subject experiments 0.4 ng/ml, or 0.36 nM of polymyxin B was required to completely inhibit neutrophil stimulation using 10 ng/ml of LPS. 90 ng/ml, or 1.58 nM BPI Protein was required for 100% inhibition of 10 ng/ml LPS. Therefore, on a molar basis the amount of BPI Protein required to inhibit LPS stimulation of neutrophils in vitro was approximately 4 times the amount required for polymyxin B. To test whether BPI Protein can inhibit LPS from other gram- negative organisms, LPS molecules with varying polysaccharide chain lengths and Lipid A were tested in the subject system (CR1 stimulation in neutrophils) against 90 ng/ml of 2X purified BPI Protein. Data shown in Table 2 demonstrate that the stimulatory dose may vary between these molecules and that LPS from both smooth and rough chemotypes as well as Lipid A are all inhibited by BPI Protein. Table 2 Effect of BPI Protein on Neutrophil Stimulation by Various Agents Inhibition of CR Upregulation on Neutrophils LPS 10 ng/ml 1 ng/ml E.COLI 0111:B4 97 * S. TYPHIMURIUM WILD TYPE 103 113 S. TYPHIMURIUM RE MUTANT 113 109 S. TYPHIMURIUM RE MUTANT 33 99 LIPID A P. AERUGINOSA 112 * * Low to no stimulation at this endotoxin concentration Example 2 Preliminary Studies On Neutrophil BPI Protein In response to LPS, human neutrophils upregulate cell surface expression of complement receptors CRl and CR3 (data not shown) . To measure this neutrophil response to LPS, freshly isolated human neutrophils were incubated with E. coli 0111:B4 LPS (data not shown) , and showed that maximal CRl upregulation is observed using 10 ng/ml LPS (data not shown) . Neutrophil stimulation with LPS was not inhibited by exogenous anti-TNF antibodies, suggesting that LPS acted directly on neutrophils in this system. BPI Protein inhibits the neutrophil response to LPS (data not shown) . Inhibition of CR upregulation was complete at a dose of approximately 1.8-3.6 nM (100-200 ng/ml) BPI Protein, compared to 0.4 nM polymyxin B required to inhibit 10 ng/ml smooth LPS (approximate m.w. 15,000) which is about 0.7 nM, matching closely with the observed value of 0.4 nM. On a molar basis, the amount of BPI Protein required to inhibit LPS was approximately 5-fold greater than the amount required for polymyxin B. BPI Protein inhibits LPS-mediated neutrophil stimulation but not stimulation by either FMLP or TNF (Table 3) . These data demonstrate that BPI Protein inhibits LPS directly and does not disrupt neutrophil mechanisms involved in CR upregulation. Table 3 Effect of BPI Protein on Neutrophil Stimulation by Various Agents Inhibition of CR Upregulation on Neutrophils Stimulus Dose % Inhibition % Inhibition CRl CR3 LPS 10 ng/ml 109 102 FMLP 10 "7 M 11 rTNF 50 U/ml Neutrophils were incubated with E. coli 0111:B4 LPS, FMLP or TNF preincubated in the presence or absence of 2.7 nM BPI Protein. Data is reported as percent inhibition of CR expression in response to each stimulus preincubated with buffer alone. Neutralization of LPS by BPI Protein occurred rapidly. Even without preincubation, both BPI Protein (and polymyxin B) inhibited more than 70% of the neutrophil response to LPS (data not shown) . Maximal inhibition was seen following only 5 minutes of preincubation. BPI Protein inhibits CR upregulation stimulated by LPS from smooth and rough bacterial strains, as well as lipid A (Table 4) . Because of the broad range of BPI Protein activity against these different forms of LPS, among which only lipid A and 2-keto-3-deoxy-octonate are shared determinants, it is likely that LPS inhibition by BPI Protein is affected through lipid A. Table 4 Inhibition of LPS and Lipid A- ■ induced Neutrophil Stimulation bv BPI Protein Inhibit:ion of CR Upregulation on Neutrophils Stimulus Dose CRl CR3 (ng/ml) % Inhibition %Inhibition None - 0 0 E. Coli 011:B4 LPS 10 100 99 S. typhimurium Wild Type LPS 10 104 100 S. typhimurium RE Mutant LPS 1 97 95 S. typhimurium RE Mutant Lipid A 1 111 104 Neutrophils were stimulated with LPS and lipid A preincubated with and without 2.7 nM purified BPI Protein. Results are expressed as percent inhibition of fluorescence intensity observed with each type of LPS alone. BPI Protein inhibits other LPS-mediated activities. At a concentration of approximately 9 nM, or 500 ng/ml, BPI Protein significantly inhibited LPS activity in the LAL assay (data not shown) . When LPS and BPI Protein were added together without preincubation, no inhibition was observed (data not shown) , indicating that BPI Protein acted on LPS, and had no effect on the LAL assay system. BPI Protein also inhibits LPS-mediated TNF production by human adherent mononuclear cells (Table 5) . Table 5 BPI Protein Inhibits LPS-lnduced TNF Production by Human Monocytes TNF (pg/ml) Produced in Response to LPS Preincubated With*: LPS Medium 100 ng/ml 500 ng/ml 250 ng/ml Buffer (ng/ml) Alone Polymyxin B BPI BPI Control 0 0 0 0 0 0 0.1 61 0 0 0 81 1 1051 96 0 0 1090 10 2053 2154 1490 1746 2325 *E. coli 0111:B4 LPS, was preincubated with BPI Protein or polymyxin B (PMB), than added to adherent peripheral blood mononuclear cells. TNF production was assayed by ELISA. BPI Protein was first purified by Elsbach and Weiss in 1978. In the initial studies, BPI Protein was isolated from azurophil granule extracts in a single step by reverse phase HPLC. Recovery of BPI Protein activity from reverse phase was poor, probably due to the denaturing conditions. Here, the purification of LPS inhibitory activity using only non- denaturing steps is shown, and it is demonstrated that most of the activity from neutrophils comigrates with BPI. Improvements in the purification have also led to very high specific activity material as will be shown in the following section. Example 3 Purification of neutrophil BPI Protein under rigorously pyrogen-free conditions A. .Materials and Methods 1. Reagents USP grade sterile irrigation water was obtained from Travenol Laboratories Inc. (Deerfield IL) ; Pyrosart filters were obtained from Sartorius GmbH (Germany) ; CM Sepharose FF was obtained from Pharmacia (Upsala, Sweden) ; Polyaspartamide weak cation exchange HPLC column (100 X 4.6mm) was obtained from the Nest Group (Southborough, MA.) ; Glycine and Bio-Sil G250 size exclusion HPLC column (600 X 7.5mm) were obtained from Bio-Rad Laboratories (Richmond, CA) ; Polyacrylamide electrophoresis gels were obtained from Novex (Encitas, CA) ; Sequencing and amino acid analysis reagents and buffers were obtained from Applied Biosystems, Inc. (Foster City, CA) ; Trifluoroacetic acid, constant boiling HCL, hydrolyzate amino acid standard, and BCA protein assay reagents were obtained from Pierce Chemical Co. (Rockford, IL) ; Limulus Amebocyte Lysate assay was obtained from Whittaker Bioproducts, Inc. (Walkersville, MD) ; Lipopolysaccharide was obtained from RIBI Immunochem Research, Inc. (Hamilton, MT) ; HPLC grade Acetonitrile from J.T. Baker (Phillipsburg, NJ) ; all other buffers and salts used were reagent grade. 18 megohm purity water was prepared by Lab Five ultrapure water system from Technic (Seattle, WA) . 0.5M NaOH for sanitization was prepared from reagent grade NaOH pellets and USP water. 2• Granule extracts from Neutrophils These were prepared as described (U.S. Serial No. 07/199,206, filed May 26, 1988) except that the percoll separation of azurophil granules was omitted. Instead, whole granule fractions were obtained by centrifuging the post nuclear supernatant at 17,000 x g for 20 minutes. The granule pellet was then suspended in a volume of l ml of 50 mM glycine, pH 2 for every 4 X 10 8 cells lysed. Resuspended granules were lysed-by five freeze/thaw cycles on dry ice ethanol followed by vigorous agitation for one hour at 4°C. The soluble extract was obtained by centrifugation at 30,000 x g for 30 minutes. 3. Limulus Amebocyte Lysate assay This assay was performed as directed by the manufacturer. Where necessary the pH of samples was adjusted to neutrality by the addition of pyrogen-free 0.5M phosphate buffer, pH 7.4, and salinity was decreased to <150 mM by dilution with USP water [Desch, C.E., et al. (1989) Infection and Immunity. 5J7:1612-1614; Friberger, P. (1985)] . The design of a reliable endotoxin test. p. 139-149. In J.W. Cape (ed.). Bacterial endotoxms: structure, biomedical significance and direction with the Limulus amebocyte lysate test. Alan R. Liss, Inc., New York; Galanos, C, et al. (1979) Chemical, physiochemical and biologic properties of bacterial lipopolysaccharides, p. 321-332. In E. Cohen (ed.) Biomedical application of the horseshoe crab (Limulidae) . Alan R. Liss, Inc., New York; Byaston, K.F. and J. Cohen (1990) J.Med. Microbiol.. 11:73-83; Tanaka, D., et al. (1982) Biochem. Biophvs. Res. Comm.. 10_5.717-723; Morita, T. , et al. (1985) J. Biochem.. £7:1611- 1620] . 4. LPS neutralization assay This assay was performed as previously described [M. Marra et al. (1990) J. Immunol. 144(21:662-6661. 5. High salt fractionation of granule extracts 200 mgs of extracted protein were pooled from various preparations and kept on ice. 1 volume of sterile 5M NaCl was added for every 4 volumes of extract. The resulting precipitate was pelleted by centrifugation at 20,000 x g for 20 minutes at 4°C. This supernatant was prepared for CM sepharose chromatography by diluting with 4 volumes of USP irrigation water and adjusting the pH with enough IM Tris pH 7.4 to give a final concentration of 50 mM. Only fresh, sterile, pyrogen-free stock salts and buffers were used. 6. CM Sepharose chromatography An XK-16 column (Pharmacia) was packed with sufficient resin to give a bed volume of 5 mis. The column was installed on a gradient FPLC equipped with a Pl pump for sample loading. Prior to use, all surfaces in contact with the mobile phase were extensively washed with 0.5M NaOH. The column was sanitized by washing at 0.2 mls/min. with 0.5M NaOH for 4 hrs. The column was then re-equilibrated and a blank run was performed. Fractions from the blank run and eluents were tested by LAL assay for pyrogenicity. Prepared extract was loaded at a flow rate of 400 mls/hr. Once loaded the column was washed with 2 to 3 column volumes of starting buffer. The granule extract was kept on ice during loading. The column was run at room temperature. 7. Weak cation exchange HPLC Weak cation exchange HPLC was performed using an Eldex ternary gradient pump equipped with a Rheodyne injector and a Gilson model 111B U.V. detector. Wettable surfaces were washed with 0.5M NaOH followed by extensive rinsing with USP water to remove all traces of base prior to installing the column. Blank fractions and eluents were tested for pyrogenicity as above. 8. Gel permeation HPLC Gel permeation HPLC was performed with the same precautions and equipment outlined for weak cation exchange HPLC, except that the TDK column was not treated with NaOH. 9. Polyacrylamide gel electrophoresis 8 to 16% acrylamide gradient gels were purchased from Novex and run according to the manufacturer's specifications. 10. Protein sequence determination An Applied Biosystems 477A pulsed liquid phase sequenator equipped with a 120A PTH amino acid analyzer was used for automated Edman degradation. 11. Microbore reverse phase HPLC Material for protein sequencing was prepared by desalting on a 30 X 2.1 mm Aquapore butyl column. Solution A was 0.1% T.F.A. in water and solution B was 70% acetonitrile in water with 0.1% T.F.A. The gradient used was 30 to 100% B in 30 minutes at a flow rate of 200 μl/minute. Detector settings were 214 nm wavelength at 2.0 absorbance units full scale. An HP 3396A was used to integrate and plot data. 12. Amino Acid Analysis Amino acid analysis was performed on the system described above using the PTC column, buffers and separation conditions provided by ABI. Sample hydrolysis and PTC derivatives were prepared using a Pico-Tag workstation from the Waters chromatography division of Millipore using the manufacturer's protocols. 13• Protein assays Protein concentrations were determined using BCA method instructions 23230, 23225 from Pierce Chemical Co. In order to minimize buffer interference, samples were diluted 10-fold and the micro reagent protocol was used. B . Results BPI Protein purified from azurophil granules was previously shown to inhibit neutrophil activation by LPS and to inhibit LPS directly in the LAL assay. In order to further define the role of BPI Protein and investigate the presence of other similar molecules in both azurophil and specific granules, the purification of LPS inhibitory activity was undertaken from whole granules extracted at acidic pH. Preliminary studies verified the presence of LPS inhibitory activity in the crude extract. To identify the endotoxin-neutralizing activity, its purification was attempted from whole granule extracts. Purification of LPS neutralizing activity was greatly enhanced by the observation that high concentrations of NaCl (IM) caused the reversible precipitation of about ninety percent of the protein present in the granule extract. Essentially all of the LPS inhibitory activity remained in the soluble supernatant. The soluble fraction was then diluted, to reduce the ionic strength, and further purified and concentrated by CM sepharose cation exchange chromatography. A broad peak of activity eluted which was subsequently further purified using a polyaspartamide high performance cation exchange column. A somewhat sharper peak of activity was recovered which co- migrated with a major protein of about 55,000 molecular weight by SDS-PAGE along with several lower molecular weight proteins. Gel permeation HPLC was used as the final purification step and identified a peak of activity which eluted with a single sharp protein peak. The purified protein migrated as two closely spaced bands on SDS-PAGE at 55,000 molecular weight representing a mixture of glycosylated and nonglycosylated species. 25% of the total endotoxin- neutralizing activity was recovered with a 250-fold purification. The purified endotoxin-neutralizing protein was subjected to reverse phase HPLC followed by N-terminal sequence analysis by automated Edman degradation. The sequence was identified as bacterial permeability increasing protein by virtue of complete homology through 39 residues. In addition the amino acid composition of the purified molecule was virtually identical to that of BPI Protein (data not shown) . To investigate whether both closely spaced bands represented BPI Protein, the purified proteins were subjected to Western blotting analysis using BPI Protein-specific rabbit polyclonal antisera raised against a synthetic peptide comprising amino acids 1-20 of BPI Protein. Both bands were immunoreactive. The differences likely arise from glycosylation. Example 4 LPS inhibitory activities of BPI Protein in vitro Purification of BPI Protein under rigorously pyrogen-free conditions, as described, supra. resulted in a more potent BPI Protein preparation. Inhibition of LPS-mediated CR upregulation was complete at 25 ng/ml BPI Protein, representing a 4-fold increase in activity compared to the material used in section I. On a molar basis this BPI Protein preparation inhibited LPS at approximately stoichiometric proportions, equivalent to molar inhibitory concentrations of polymyxin B. BPI Protein also inhibited LPS-mediated TNF production by human adherent mononuclear cells at a lower concentration following purification under pyrogen-free conditions (Tables 7 and 8) . BPI Protein binds to LPS (data not shown) . In these experiments, 4 μg of LPS/well was immobilized on 96 well plastic plates, then incubated with varying concentrations of BPI Protein, and developed with anti-BPI Protein polyclonal antisera. BPI Protein binding to LPS was inhibited by polymyxin B (data not shown) , demonstrating specificity of BPI Protein binding. BPI Protein binds to LPS in the presence of both plasma (data not shown) and serum (data not shown) , demonstrating potential in vivo efficacy of BPI Protein. Table 7 BPI Protein Inhibits LPS-Induced TNF Production by Human Monocytes TNF (pg/ml) Produced in Response to LPS Preincubated with*: LPS Medium 100 ng/ml 400 ng/ml 150 ng/ml 25 ng/ml Buffer ng/ml alone PMB BPI BPI BPI Control 0 0 0 0 0 0 0 0.1 98 79 0 0 0 269 1 1150 1207 0 0 0 1292 10 1370 1270 145 353 559 1413 *E. coli 0111:B4 LPS. was preincubated with BPI Protein or polymyxin B (PMB), then added to adherent peripheral blood mononuclear cells. TNF ' production was assayed by ELISA. Table 8 Inhibition of LPS-Induced THF Production by Human Monocytes TNF (pg/ml) Produced in Response to LPS Preincubated with*: 1000 ng/ml 100 ng/ml 250 ng/ml 50 ng/ml 10ng/ml Buffer LPS Polymyxin B Polymyxin B BPI BPI BPI Control 10 333+18 601+257 270+23 270+67 436+38 697+37 100 769+101 1140+73 834+30 686+84 1005+50 892+47 1000 844+144 1016+20 1130+10 778+189 1025+71 723+88 1685+121 1541+397 1558+139 1268+374 1554+324 1423+447 *BPI Protein or polymyxin B sulfate were preincubated with 0-10 ng/ml E. coli 0111:B4 LPS or 0.1 X w/v killed S. aureus then added adherent peripheral blood mononuclear cells. TNF production was assayed by ELISA, and is reported as ng/ml. Example 5 Inhibition of LPS-induced TNF production by neutrophil and recombinant CHO cell-derived BPI Protein Human peripheral blood mononuclear cells were isolated on Ficoll-Pague (Pharmacia) gradients, washed 2X in pyrogen-free HBSS (Hazelton) , and resuspended at 5 X 10 6 /ml in RPMI (Gibco) media without serum. Two hundred μl of this cell suspension was incubated in each well of flat-bottom 96-well tissue culture dishes (Costar) for 2 hours at 37°C. Nonadherent cells were removed by washing 2X with RPMI + 10% autologous heat inactivated serum. Adherent mononuclear cells were stimulated with E. coli 0111:B4 LPS which had been preincubated for 30 minutes at 37°C with buffer, BPI Protein or polymyxin B (Gibco; 7900 U/ml) . Supernatants were harvested four hours after LPS mixtures were added. Secretion of TNFα was quantitated by ELISA (Endogen) (results at Table 9) . Table 9 Inhibition of LPS -Induced 1NF Production bv Human Monocvt :es 10 nσ/ml LPS 1 nσ/ml LPS 0 LPS BPI (nM) : 0 7.3 1.4 0.3 0 7.3 1.4 0.3 Lot No. 78038n 626 129 159 203 334 153 187 165 113 148104n 98 104 162 98 119 162 148113n 92 114 151 71 129 155 148159r 82 158 155 87 136 147 148165r 124 128 138 116 129 146 148179r 85 139 134 93 131 166 n=natural r=recombinant Normal anesthetized mice were challenged by the intranasal route with 10 ng/ml E. coli 0111:B4 LPS (results in Table 10) . Twenty minutes before challenge, anesthetized mice were treated by the intranasal route with 50μl saline, BPI Protein or polymyxin B solution. At one hour after LPS challenge, mice were re-anesthetized, and 0.7 ml of saline containing 1% human serum albumin was added to the lungs via the trachea. The lungs were gently kneaded. A 0.5 ml volume broncheo- alveolar lavage (BAL) fluid was aspirated, cells were pelleted by centrifugation, and the BAL sample was stored at -70°C. The TNFα level in the BAL fluid was determined by measuring cytotoxicity towards WEHI clone 13 mouse fibrosarcoma cells and is summarized in Table 10. Human rTNFα (Chiron) was used as the standard. Table 10 Inhibition of TNFα Production as Measured in Broncheoalveolar Lavaσe Fluid Mouse Saline BPI Polymyxin B Control 0.86μg l.Oμg (15 pmol) (782 pmol) 1 1200 15 74 2 675 63 50 3 5560 425 132 4 2800 67 370 5 5250 1310 640 Mean ± SD 3097 ± 2250 376 ± 547 253 ± 251 R_raτn 1*_ 6 Expression of recombinant CHO cell-derived BPI Protein Recombinant CHO cell-derived BPI Protein was obtained as described in the following example. In order to produce BPI Protein and/or BPI Protein variants in mammalian cells, the cDNA sequences must be inserted into a suitable plasmid vector. A schematic drawing of cDNA encoding BPI Protein is shown in Figure 1. A suitable vector for such an application is pSV-1, which contains the origin of replication and early and late promoters of SV40, followed by multiple insert cloning sites, followed by the termination sequences from the hepatitis B surface antigen gene (Figure 5) . Also contained within the plasmid are an origin of bacterial DNA replication, and the genes encoding ampicillin resistance and dihydrofolate reductase. Similar vectors have been used to express other foreign genes (McGrogan, et. al., Biotechnology 6, 172-177). Vector DNA was prepared for acceptance of BPI Protein cDNA sequences by digestion with Hindlll and BamHI, and dephosphorylation with alkaline phosphatase. Several BPI Protein and BPI variant cDNA-containing inserts were prepared for insertion into pSV-1. First, an insert encoding BPI Protein was prepared by digestion of the parent plasmid with appropriate restriction enzymes, for example, EcoRI and Bglll, yielding two DNA fragments containing portions of the BPI Protein coding sequence. These two fragments were ligated together into prepared SV-1, and the recombinant clones obtained were screened by restriction enzyme digestion for the presence of the two inserts in the proper orientation. Two cDNAs encoding truncated forms of BPI Protein were generated using oligonucleotide-directed DNA amplification of the parent BPI Protein insert DNA. The amplifying oligos were designed to replace codons 212 (oligo 459) . (Figure 2) and 337 (oligo 460) (Figure 3) with stop codons, in addition to a BamHI cloning site. At the 5' -end of both constructs, oligo 458 was used in the amplifica ions to create a Hindlll site immediately upstream of the translational start codon ATG (Figure 4) . Thus, three BPI- variant encoding inserts were created, each encoding 55 kDa, 38 kDa, and 25 kDa portions of BPI Protein, and each was ligated separately into prepared vector DNA. Figures 9 and 10 show the amino acid sequences of the 38 kDa and 25 kDa portions, respectively. Each of the three constructs was verified by restriction digest analysis, and then prepared in amounts sufficient for transfection into CHO cell line DUXB11 cells. Transfection was performed using lipofectin, and the resulting transformed cells were selected in the presence of increasing amounts of methotrexate using protocols which are standard in the art. The transformed cells were cultured under conditions standard in the art, and supernatants from either transfected pools or clones derived from the pools were assayed for BPI Protein or BPI variant by the BPI Protein ELISA method discussed infra. Figure 7 shows SDS-PAGE analysis of native BPI Protein, and two lots of CHO-derived recombinant BPI Protein. Recombinant BPI Protein migrates as a single band on an SDS-PAGE gel at about 55 kDa. Recombinant BPI Proteins produced in CHO cells exhibit a slightly altered migration pattern on SDS-PAGE from native protein, indicating that the molecule may also be processed differently in CHO cells than in neutrophils or HL60 cells. Such processing may be either responsible for, or a result of, the molecule being secreted rather than packaged into granule membranes. Amino acid analyses of recombinant BPI and native BPI are shown in Table 11 which shows the two to be essentially the same. Table 11 Amino Acid Composition of recombinant BPI 148179 recombinant native D+N 27 38 E+Q 35 39 S 47 44 G 31 26 H 12 14 R 17 12 T 21 21 A 26 25 P 33 32 Y 16 14 V 33 37 M 10 15 I 26 26 L 50 47 F 25 25 K 42 36 total 451 451 (C, not quantitated) Example 7 Expression of τ<=-r_" binant BPI Protein in insect cells 1. construction of plasmid expression vector In order to produce BPI Protein and/or BPI Protein variants in insect cells, the cDNA sequence must first be inserted into a suitable plasmid expression vector, such as pAC373 (see Figures 6-1 and 6-2) . Appropriate restriction sites for this insertion were created by standard site-directed mutagenesis procedures. The essential properties of a suitable expression vector include a transcriptional promoter such as the polyhedron gene promoter of pAC373, and flanking homologous sequences to direct recombination into the baculovirus genome. A polyadenylation signal, such as the one from the polyhedron gene present in this plasmid vector, may or may not be necessary for expression of the recombinant gene. A marker gene such as the beta-galactosidase gene of E. coli. juxtaposed to regulatory sequences including a transcriptional promoter and possibly a polyadenylation signal, may be included in the vector but is not essential for Bactericidal/Permeability Increasing Protein expression. A typical vector for such purposes, pAC373, is shown in Figures 6-1 and 6-2. 2. Creation of recombinant baculovirus A chimeric baculovirus was created by homologous recombination between the expression plasmid containing the BPI Protein target gene (or truncations thereof derived as described supra) and wild type baculovirus DNA. Plasmid and wild type baculovirus DNA were co-precipitated by the calcium phosphate technique and added to uninfected Spodoptera fruσiperda (Sf9) insect cells. Four to seven days following transfection, cells exhibited a cytopathic morphology and contained the nuclear occlusion bodies typically produced by viral infection. The cell-free culture media containing both wild type and recombinant virus was harvested and assayed for BPI Protein activity. 3. Identification and isolation of chimeric baculovirus Clonal isolates of virus was obtained from this co- transfection stock by plaque purification on Sf9 cell monolayers overlaid with agarose. Candidate plaques for analysis will be identified by a plaque morphology negative for occlusion bodies. If the expression plasmid contains a marker gene such as beta galactosidase, recombinant plaques will be indicated by the blue color produced from a chromogenic substrate suchas 5-bromo-4-chloryl-3-indolyl-__.-D- galactopyranoside (x-gal) in the agarose plating medium. Picked plaques will be used for inoculation of cells in multi- well dishes. The resulting cell lysates and infected cell supernatants can be evaluated for expression of recombinant BPI Protein using standard activity or immunological assays. Positive wells may require additional rounds of plaque purification to obtain pure recombinant virus stocks free from wild type contamination. 4. Batch production of BPI Protein Sf9 cells were adapted to growth in serum-free, low protein medium such as ExCell (J.R. Scientific) . Cells were collected from suspension culture by gentle centrifugation and resuspended in fresh medium containing the viral inoculum at a concentration of ten million cells per ml, using a multiplicity of infection of one virus plaque-forming unit per cell. After a period of two hours, the culture was diluted five-fold with fresh medium and incubated for two to three days. At the end of that time, the cells were pelleted by centrifugation and the conditioned medium was harvested. BPI Protein was purified from the cell-free supernatant by standard means. 5. Characterization of insect cell derived BPI Protein BPI Protein produced in insect cells using a baculovirus expression system is a glycosylated protein of approximate molecular weight of 55,000 kd. The N-terminal amino acid sequence is identical to that of mature mammalian cell- produced BPI Protein, indicating correct processing of the signal sequence. The specific activity of endotoxin binding of recombinant protein was indistinguishable from BPI Protein obtained from natural sources. Rvampl _ . ft Expression of recombinant BPI Protein with signal sequence in E. Coli A. Construction of pT7BPI protein plasmids Oligonucleotides were prepared on an Applied Biosystems 380B DNA synthesizer for use in oligonucleotide-directed DNA amplification. The oligonucleotides were created with Ndell and BamHI restriction sites at the 5' and 3' ends, respectively, of the BPI Protein DNA. In addition, another oligonucleotide containing a BamHI restriction site was used to create the truncated proline-212 version of the BPI Protein DNA. Following the amplification reactions, fragments were purified and digested with Ndel and BamHI. The plasmid pGEMEX-l (available from Promega) was selected as the vector for the constructions. pGEMEX-l contains a T7 promoter which can be used for the expression of downstream sequences when placed into the proper host. The vector was cleaved with BamHI and, following purification, partially digested with Ndel to generate a vector with a single Ndel site and a single BamHI site. The fragments were ligated and transformed into the IL . coli strain M101 using the Hanahan transformation protocol. The transformed bacteria were plated on LB plates containing carbamicillin and incubated overnight at 37°C. Resistant colonies were selected and analyzed by preparing mini-plasmid preparations and digesting with the appropriate restriction enzymes. Digests were analyzed on both 1% agarose gels and 5% polyacrylamide gels. The expression host, E. coli strain JM109(DE3), was transformed using 1 μl of the mini-plasmid preparation and the Hanahan transformation protocol (DNA Cloninσ; A Practical Approac . Vol I, D.M. Glover Ed., IRC Pr. Ltd., Chapter 6, pp. 109-135 (1985)). JM109(DE3) contains a chromosomal copy of the gene for T7 RNA polymerase which can be induced with IPTG. The transformed bacteria were plated on LB plates containing carbamicillin and incubated overnight at 37°C. Results are not shown. B. Results When a signal sequence is operably linked to the mature BPI Protein cDNA sequence in the expression plasmid as provided in a full length clone and reported by Gray, et al. [Journ. of Biol. Chem., 264:9505 (1989)], no bacterial colonies are obtained, whereas numerous colonies can be obtained if the signal sequence is deleted. Since the full-length and proline-212 truncated forms of BPI Protein containing the signal peptide do not give colonies while those forms that do not contain the signal peptide do give colonies, the BPI Protein was expressed in an active form and is processed correctly, sending the protein to the periplasmic space of the bacteria (the location in bacteria to which proteins possessing a signal peptide are sent) where the bactericidal activity kills the cell. This also implies that both the full-length form and the proline-212 truncated form are active and capable of bactericidal activity. Example 9 Expression of recombinant E. Coli-derived BPI Protein 1. Generation of BPI Protein expression vector To generate a construct for E. coli expression, the BPI Protein encoding sequence was subcloned from the CHO cell expression vector by PCR using the oligonucleotides: BPI.forward R • C . AC.C.. GCC.GTG-ACA.CA .ATG.GTC. AAC.CCT.GGC.GTC BPI.reverse 5'GAC.GTT.CTC.TAT.AAA. TGA. GG.ATC.CTA.GCT.C generating a Ndel and BamHI site respectively. The resulting fragment was cut with Ndel and BamHI and subcloned into pGEMEX-l VECTOR (Promega) . The pGEMEX E. coli expression vector contains three RNA polymerase fragments. Downstream from gene 10 is a multiple cloning sequence. In-frame subcloning can be performed to generate fusion proteins, if the protein of interest has the appropriate restriction sites in-frame. To avoid the problems associated with fusion protein production, the gene 10 region was removed from pGEMEX, but the T7 RNA polymerase binding site and the Ndel and BamHI cloning sites were retained. To accomplish this the Ndel site at 3251 was removed by partial digest followed by Klenow fill- in and re-ligation. This plasmid is referred to as pT7BPI. The correct sequence was confirmed by sequencing the entire coding region for BPI Protein but with the leader sequence deleted, such that the first amino acid coded for by the plasmid is methionine. 2. Mutaσenesis for Construction of BPI variants The plasmid pT7BPI has an fl ori for the production of single- stranded DNA. This pT7BPI was transformed into the E. coli strain CJ236 for the production of ssDNA to be used as a template for site-directed mutagenesis according to the method of Kunkel [(Kunkel, T.A. (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., Lilley, D.M.J. Eds.) Vol.2, p. 124, Springer-Verlag, Berlin and Heidelberg] . Mutagenic oligonucleotides were generated with homology on the 5' side and 12 nucleotides of exact homology on the 3' side of the mismatched codon or codons corresponding to the site of mutation. The mutagenic oligonucleotide was purified using OPC columns as described by ABI, phosphorylated by standard enzymatic techniques, annealed to ss-pT7BPI generated in CJ236 cells, extended at 37°C with T7DNA polymerase and ligated with 5 T4 ligase. The resultant closed circular ds-DNA was transformed according to standard protocols (see, e.g., Sambrook, et al.) into RecA+ E. coli such as JM101 and plated. Single colonies were grown for production on ds-DNA by standard techniques for double-stranded sequencing. Clones 0 with the correct mutation were transformed into JM109 (DE3) for expression. 3. Expression of BPI Protein JM109 (DE3) contains a chromosomal copy of the gene which 5 codes for T7RNA polymerase under the control of the inducible lac promoter. JM109 (DE3) containing pTBPI (or a variant of BPI) is grown overnight in 2xYT media (BiolOl, La Jolla, CA) + 0.2% glucose + lOOmg/ml carbenicillin at 26-37°C. Low temperature and high nutrient-containing solution is helpful 0 in generating productive inoculants. The inoculum is diluted 1:250 to 1:500 and grown to OD^o,,,,, _= 1 in a shake flask or ODeoo nn , ≤ 50 in a fermentor at 26-37°C, and induced with IPTG at ^ 0.1-1.0 mM for 4-24 hours. The bacteria are collected by centrifugation, resuspended in 10 mM TRIS, pH 8, l mM EDTA, 5 and disrupted by high pressure homogenization. When bacteria were grown at 37°C as is standard in the art, essentially all of the BPI Protein was produced insoluble in inclusion bodies. However, when the temperature was lowered below 37°C, increasing amounts of BPI Protein were recovered in soluble, 0 active form. Results 1. Expression of insoluble BPI Protein in E. coli Expression at 37°C resulted predominantly in the production of BPI Protein in insoluble inclusion bodies, as identified by light microscopy. E. coli cells were lysed in Tris pH ' 6.0 with an Ultra-Turrax homogenizer. Inclusion bodies were 5 collected by centrifugation, washed with 1 M NaCl, 0.05% triethylamine, and the protein refolded by rapid dilution from a 6M guanidine solution into various buffers. BPI Protein was purified by a capture of Fast S Sepharose and eluted with 0.6 M NaCl, diluted to 0.25 M NaCl and passed over Fast Q 0 Sepharose to remove endotoxin and recaptured on Fast S Sepharose and eluted by 0.6 M NaCl or a gradient of 0.25 to 1 M NaCl. Refolding under a variety of conditions resulted in active BPI Protein with approximately 0.1% yield from all BPI (i.e., lmg/g total BPI). 5 2. Expression of soluble active BPI Protein in E. coli Alternatively, it was discovered that BPI Protein can be generated in a soluble form within E. coli by adjusting the fermentation conditions. A greater yield of soluble BPI 0 Protein was discovered as the fermentation temperature is decreased from 37°C to 26°C with a concomitant loss in inclusion body material. This was demonstrated in shake flask _ cultures grown in 2xYT media (BiolOl, La Jolla, CA) + 0.2% glucose + lOOmg/ml carbenicillin. The ability to produce BPI 5 Protein is quite unpredicted, since BPI Protein is bactericidal for E. coli and BPI Protein constructs with a leader sequence designed to secrete BPI Protein into periplasmic space resulted in cell death. However, it is shown here that BPI Protein not secreted but retained in the 0 cytoplasm can be maintained in high concentrations without cell death. To purify soluble BPI Protein, the cell lysate from the disruption step (disruption by microfluidizer) was clarified by centrifugation and filtration. followed by centrifugation and filtration, and the soluble protein purified as described below. Alternatively, cell lysate can be clarified by treatment with polycations such as polyethyleneimine or Biocryl (Toso Haas) . The generation of soluble material has many advantages, i.e., there are no refolding steps and there is greater reproducibility from batch to batch. BPI Protein was produced with yields corresponding to about l mg per liter of production at a cell density of 1 OD,^^. By using standard fed-batch fermentation techniques, the overall production can be enhanced significantly to achieve cell densities of greater than 50 OD^o ^ ,,. One example would be to inoculate the bacteria in a media consisting of 8 g per liter tryptone, 5 g per liter yeast extract, 2.5 g per liter NaCl, 2 g per liter glucose, and 50 microgram per ml carbenicillin, with a feed of 100 g per liter tryptone, 60g per liter yeast extract, 200 g per liter glucose and 0.1 g per liter carbenicillin and a maintained pH of 7.1 and dissolved oxygen at 50%. This E. coli synthesis of a protein toxic to E. coli represents a substantial advance in the state of the art of BPI Protein production. Fermentation conditions can be varied by those skilled in the art to maximize production. Further advances to increase the amount of soluble BPI Protein include overexpression of cellular cofactors or chaperon proteins that are necessary or advantageous to BPI Protein folding in the cell. Also, one could optimize the amino acid structure of BPI Protein, while maintaining its biological activity, so as to produce an active BPI Protein variant which is expressed at a high level in bacteria. BPI Protein produced in Chinese hamster ovary cells migrate with a higher molecular mass on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) than E. coli- produced BPI Protein, indicating that these molecules are processed differently in mammalian cells than in E. coli (Figure 11) . 3. Purification of E coli-derived BPI Protein The purification of BPI Protein from E. coli lysates presents several obstacles to be overcome. BPI Protein is a potent endotoxin-binding molecule, and experimental evidence has shown there to be more than 100 million endotoxin units per milliliter in the E. coli cell lysate (based on the limulus amebocyte lysate assay) . Thus the BPI Protein produced in E. coli is complexed to endotoxin and associated cell wall fragments. To obtain purified soluble active BPI Protein from E. coli. the BPI Protein must be purified away from its endotoxin while retaining its activity. These difficulties have now been overcome with a combination of steps designed to produce highly purified, soluble, active, endotoxin-free BPI Protein from E. coli. . Purification Process A E. coli bacteria expressing BPI Protein were lysed by high pressure homogenization (Microfluidics, Newton, MA) in lOmM Tris ImM EDTA pH 8. Centrifugation and filtration were used to remove remaining whole cells and cell debris. MgCl 2 was added to 500mM, and incubated for 30 minutes at 37°C. Acetic acid was added to lower pH to 4.0. Additional incubation, 30 minutes at 37°C, caused precipitation of much of the contaminating protein, lipid, and DNA. After centrifugation and filtration, BPI Protein was left soluble in the supernatant. It was diluted 3x and bound to a cation exchanger, e.g., Poros HS (Perseptive Biosystems, Cambridge, MA) , Fast S Sepharose, or CM Sepharose (Pharmacia, Piscataway, NJ) . A series of washes followed: pH 6.5, 8.5 and 10.0. The pH was brought back to 6 using a suitable buffer, and elution was achieved with a 0-2M NaCl gradient or step elution. The pH of the eluant was raised to 8.5, and the solution was passed through a suitable anion exchanger, e.g. Poros HQ, Fast Q Sepharose, or DEAE Sepharose, to remove any remaining endotoxin and DNA, as well as contaminating anionic compounds. BPI Protein does not bind under these conditions. BPI Protein was then bound to another cation exchange column, and eluted with either a gradient or a step of NaCl. The N-terminal sequence of BPI Protein was confirmed by automated Edman degradation and activity verified by inhibition in the Limulus amebocyte lysate assay (Figure 13) . 5. Purification Process B BPI Protein complexed with endotoxin, as it is in the cell lysate, will not bind to cation exchange. Thus, as an alternative purification scheme, the cell lysate can be passed over a cation exchange column, such as Poros HS (Perseptive Biosystems Cambridge, MA) , Fast S Sepharose, or CM Sepharose (Pharmacia, Piscataway, NJ) , at the lowest possible pH at which the BPI Protein-endotoxin complex will not bind. This removes many of the contaminating cationic entities which would otherwise co-elute with BPI Protein in a subsequent cation exchange step. The column flow through is then brought to 500mM MgCl 2ι incubated 30 minutes at 37°C and pH adjusted to 4.0 with acetic acid. After centrifugation and filtration, BPI Protein is soluble in the supernatant. BPI Protein is then diluted 3x and bound to a cation exchanger, e.g., Poros HS (Perseptive Biosystems, Cambridge, MA) , Fast S Sepharose, or CM Sepharose (Pharmacia, Piscataway, NJ) . A series of washes would follow: pH 6.5, 8.5 and 10.0. The pH is then brought back to 6 in a suitable buffer, and elution achieved with a 0-2M NaCl gradient or step elution. The pH of the eluant is then raised to 8.5, and the solution passed through a suitable anion exchanger, e.g., Poros HQ, Fast Q Sepharose, or DEAE Sepharose to remove any remaining endotoxin and DNA, as well as contaminating anionic compounds. BPI Protein does not bind under these conditions. BPI Protein is then bound to another cation exchange column, and eluted with either a gradient or a step of NaCl. BPI Protein is then formulated in lOmM Sodium Succinate, llOmM NaCl, pH 6.0 by size exclusion chromatography using Sepharose CL-6B. 6. Purification Process C An alternative lysis protocol would involve using additives that would prevent the formation of BPI Protein-endotoxin complexes. Magnesium Chloride would be added to the cell suspension prior to lysis, with pH at either 8 or 4. Lysis would be achieved by high pressure homogenization. After centrifugation and filtration, BPI Protein would be soluble in the supernatant. BPI Protein would be diluted 3x and bound to a cation exchanger, e.g., Poros HS (Perseptive Biosystems, Cambridge, MA) , Fast S Sepharose, or CM Sepharose(Pharmacia, Piscataway, NJ) . A series of washes would follow: pH 6.5, 8.5, 10.0. The pH would be brought back to 6 in a suitable buffer, and elution achieved with a 0-2M NaCl gradient or step elution. The pH of the eluant would be raised to 8.5, and the solution passed through a suitable anion exchanger, e.g. Poros HQ, Fast Q Sepharose, or DEAE Sepharose to remove any remaining endotoxin and DNA, as well as contaminating anionic compounds. BPI Protein does not bind under these conditions. BPI Protein would then be bound to another cation exchange column, and eluted with either a gradient or a step of NaCl. BPI Protein would then be formulated in lOmM Sodium Succinate, llOmM NaCl, pH 6.0 by size exclusion chromatography using Sepharose CL-6B. 7. Use of endotoxin removal aσents Endotoxin-removing polycations such as polyethylimine or BioCryl (Toso Haas) can be added to cells which are next lysed by high pressure homogenization. After centrifugation and filtration, BPI Protein would be soluble in the supernatant. BPI Protein would be bound to a cation exchanger, e.g. Poros HS (Perseptive Biosystems, Cambridge, MA) , Fast S Sepharose, or CM Sepharose (Pharmacia, Piscataway, NJ) . A series of washes would follow: pH 6.5, 8.2 and 10.0. The pH would be brought back to 6 in a suitable buffer, and elution achieved with a 0-2M NaCl gradient or step elution. The pH of the eluant would be raised to 8.5, and the solution passed through a suitable anion exchanger, e.g., Poros HQ, Fast Q Sepharose, or DEAE Sepharose to remove any remaining endotoxin and DNA, as well as contaminating anionic compounds, BPI Protein does not bind under these conditions. BPI Protein would then be bound to another cation exchange column, and eluted with either a gradient or a step of NaCl. BPI Protein would then be formulated in lOmM Sodium Succinate llOmM NaCl pH 6.0 by size exclusion chromatography using Sepharose CL-6B. 8. Removal of endotoxin from the outer membrane prior to cell lysis Alternatively, the outer membrane could be removed from the bacteria prior to lysis, thus removing the endotoxin and the need for subsequent endotoxin removal in the purification. An example would be to add bivalent chelating reagents such as EDTA or EGTA or by adding a detergent such as Triton X-100 [S.T.L. Harrison, Biotech. Adv. 9:217-240 (1991)]. 9. Removal of endotoxin from BPI Protein by chromatoσraphic processes Endotoxin can be removed from BPI Protein by passing BPI Protein through an anion exchanger, e.g., Poros HQ, Fast Q Sepharose, or DEAE Sepharose, at a high pH (8.5 or above), or by binding BPI Protein to a cation exchanger, e.g., Poros HS (Perseptive Biosystems, Cambridge, MA) , Fast S Sepharose, or CM Sepharose (Pharmacia, Piscataway, NJ) , and washing extensively at high pH prior to elution with NaCl. The BPI Protein-expressing E. coli could be suspended in low pH buffer, e.g., 20mM Sodium Acetate pH 4.0, prior to lysis by high pressure homogenization. Alternatively, the pH could be lowered after lysis. After centrifugation and filtration, BPI Protein would be soluble in the supernatant. BPI Protein would be bound to a cation exchanger, e.g., Poros HS (Perseptive Biosystems, Cambridge, MA) , Fast S Sepharose, or CM Sepharose (Pharmacia, Piscataway, NJ) . A series of washes would follow: pH 6.5, 8.5 and 10.0. The pH would be brought back to 6 in a suitable buffer, and elution achieved with a 0- 2M NaCl gradient or step elution. The pH of the eluant would be raised to 8.5, and the solution passed through a suitable anion exchanger, e.g., Poros HQ, Fast Q Sepharose, or DEAE Sepharose to remove any remaining endotoxin and DNA, as well as contaminating anionic compounds. BPI Protein does not bind under these conditions. BPI Protein would then be bound to another cation exchange column, and eluted with either a gradient or step of NaCl. BPI Protein would then be formulated in lOmM Sodium Succinate, llOmM NaCl, pH 6.0 by size exclusion chromatography using Sepharose CL-6B. 10. Activity Assay of BPI Protein BPI Protein produced in soluble form at low temperature in E. coli and purified as described above in purification process A was tested for ability to neutralize endotoxin in the limulus amebocyte lysate (LAL) assay. This assay is based on the ability of gram-negative bacterial endotoxin to catalyze the activation of a pro-enzyme in the LAL. The initial rate of activation is determined by the rate of color formation using the substrate Ac-Ile-Glu-Gly-Arg-pNA and monitoring the absorbance at 405nm photometrically after the reaction is stopped. There is a linear correlation between absorbance at 405 nm and endotoxin concentration between 0.1 and 1 EU/ml. Accordingly, 1EU of endotoxin was mixed with a dilution series of E. coli BPI Protein in a microtiter plate at 37°C followed by substrate addition. The absorbance in each well of the plate was measured using a Molecular Devices plate reader. Figure 13 shows the inhibition of LAL activation by E. coli BPI Protein, with an IC 50 = 0.45 mg/ml. Thus, E. coli-derived BPI Protein is able to bind and neutralize endotoxin. BPI Protein produced in soluble form at low temperature in E. coli and purified as described above in purification process A was tested for inhibition of endotoxin-mediated TNF induction by human adherent mononuclear cells. Blood collected in acid citrate dextrose containing vacutainer tubes (Becton Dickinson, Rutherford, NJ) was diluted in Hank's balanced salt solution (HBSS) minus Ca +2 and Mg +2 . Mononuclear cells were separated using Ficol-Paque (Pharmacia Inc., Piscataway, N.J.), collected and washed three times in HBSS, and the proportion of monocytes was estimated by microscopic examination. Cells were brought up to an appropriate volume of RPMI 1640 with glutamine and antibiotics and without serum to give approximately 1 x 10° monocytes/ml. Cells were plated into 96 11 flat bottom tissue culture plates (Costar, Cambridge, MA) , 200 μl/well, and incubated for two hours at 37°C in a humidified incubator with 7% 0 2 . Cells were then washed three times in warm RPMI 1640 without serum. After the last wash was aspirated, 200 μl/well RPMI 1640 with 10% autologous heat-inactivated serum was added. To each well was then added the 22μl of 10X solution of E. coli endotoxin preincubated in buffer, polymyxin B, or BPI Protein. Cells were incubated with the endotoxin mixture for four hours at 37°C, then the supernatants were collected and assayed for TNFα antigen by ELISA (Endogen, Inc., Boston, MA) (Figure 12) . 11. BPI Protein ELISA A. Materials Immulon-4 96 well plates (Dynatech Labs, 011-010-3750); 12- channel 50-200μl pipettor; P20, P200, P1000 pipettors; micropipette and multichannel pipette tips; reagent reservoirs (Costar) ; racked 1 ml tubes (BioRad) ; and polypropylene 15 ml conical tubes. B. Reaσents Coatinσ Buffer: 25 mM Sodium Borate pH 9.5. Blockinσ Buffer: 5% BSA (Sigma A-7030) in PBS. ELISA Buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 1 mg/ml BSA (Sigma A-7030) , 0.05% Tween 20 (Sigma, P-1379) , 1 μg/ml Polymyxin B Sulfate (Gibco/BRL, 7900 U/mg) . Standard and sample diluent = Wash/Sample Buffer with 60 mM MgCl 2 or appropriate solution for unknowns (e.g. if testing tissue culture supernatants, use REM + dFBS) . Substrate Buffer: 24.5 mg MgCl 2 (make 500 ml), 48 ml Diethanolamine, bring up to ~ 400 ml with Lab V H 2 0, adjust to pH 9.8, bring up to 500 ml with Lab V H 2 0. Antibodies: INV 1-2 IgG (protein A purified Rb anti BPI antibody, 5 mg/ml) ; and biotinylated anti-BPI antibody. Also, monoclonal antibody HA-1A may be used. Anti-BPI antibodies may be produced, for example, by immunizing a rabbit with BPI, and recovering the anti-BPI antibodies produced thereby using methods known to those skilled in the art. Other Reaσents: BPI standard (Lot 149713 = 1.0 mg/ml aliquots stored at -70°C) ; PNPP substrate tablets (5 mg/tablet: Sigma 104-105); Streptavidin-Alkaline Phosphatase (BioRad, 170-3554, Lot 72439) . BPI may be obtained, for example, according to the method described at column 11, line 62 through column 12, line 13 of U.S. Patent No. 5,089,274. C. Procedure Coatinσ Plates: Note: Coat plates up to 1 month in advance. Store plates at 4°C until needed. Dilute anti-BPI IgG antibody (protein A-purified) to 5 μg/ml in 25mM Na Borate, pH 9.5 (10 ml/plate) . Add 100 μl to each well of 96-well plate (Immulon-4) . Incubate overnight at 37°C. Refrigerate until used. Blockinσ Plates: Flick coating antibody out of plates and blot plate on paper towels. Add 200 μl 5% BSA in PBS to each well. Incubate 1-4 hours at 37°C or overnight at 4°C. Flick out blocking solution, wash 3X with ELISA buffer and blot plate on paper towels. BPI Standards and Unknowns: Note - BPI standards and samples should be diluted with ELISA buffer/60mM MgCl 2 and only in propylene tubes! Thaw new standard aliquot (0.5 ml at 1 mg/ml) every 2 months. Make stock solution of purified BPI at 100 ng/ml by: a) diluting 149713 BPI 5 μl + 495 μl diluent (= 1:100; 10 μg/ml), then again 1:100 for a final concentration of 100 ng/ml in l ml (this is enough 100 ng/ml stock solution for 2 standard curves in triplicate) ; and b) making 1000 μl of each of the following standard concentrations as follows: Final TBPIl 100 nσ/ml BPI buffer (μl) nσ/ml (μl) 15 150 850 5 50 950 1 20 1980 0.50 0 500 + 500 of lng/ml 0.25 0 750 + 250 of lng/ml 0.10 0 900 + 100 of lng/ml 0.05 0 9500 + 50 of lng/ml 0 0 1000 Add 100 μl/well standards and unknowns and incubate at 37°C for 2 hours. Wash 4 X with Sample/Wash Buffer and vigorously blot on paper towels. 2nd Antibody (Coniuσate) : After final wash, blot plate vigorously, add 100 μl of biotinylated anti-BPI Ab at 1:2000 (=5μl in 10ml of wash/sample buffer) to each well. Incubate at 37°C for 1 hour, or overnight at 4°C. Wash 4 X and blot dry. Streptavidin Alkaline Phosphatase: After final wash, blot plate vigorously, add 100 μl 1:1000 (=10μl in 10ml) Steptavidin-Alkaline Phosphatase conjugate to each well. Incubate at 37°C for 45 minutes, or overnight at 4°C. Wash 4 X and blot dry. Substrate: After final wash, blot plate vigorously, and add 100 μl substrate solution (2 X 5 mg PNPP substrate tablet/10 ml substrate buffer) . Note: Prepare fresh substrate immediately before use by dissolving 2 tablets/lOml substrate buffer. Read plate at 405nm after developing for 25-35 min. Keep plate in the dark between readings.

Claims: What is claimed is: 1. A method for producing non-glycosylated Bactericidal/ Permeability Increasing Protein using a gram-negative bacterial cell, which comprises transfecting the cell with a vector encoding Bactericidal/Permeability Increasing Protein; culturing the cell so transfected under conditions permitting the production of non- glycosylatedBactericidal/PermeabilityIncreasingProtein in the cell; and recovering from the cell the non- glycosylated, Bactericidal/Permeability Increasing Protein so produced. 2. The method of claim 1, wherein the Bactericidal/ Permeability Increasing Protein has no leader peptide attached thereto when expressed. 3. The method of claim 1, wherein the conditions permitting the production of non-glycosylated Bactericidal/ Permeability Increasing Protein in the cell comprise low temperature. 4. The method of claim 1, wherein the recovering comprises the steps of (a) lysing the cell under conditions preventing the formation of a complex between the Bactericidal/Permeability Increasing Protein and LPS so as to release the Bactericidal/Permeability Increasing Protein from the cell; and (b) isolating the Bactericidal/Permeability Increasing Protein so released. The method of claim 1, wherein the recovering comprises the steps of (a) lysing the cell under conditions permitting the formation of a complex between the Bactericidal/Permeability Increasing Protein and LPS; (b) isolating the resulting Bactericidal/Permeability Increasing Protein-LPS complex; (c) dissociating the Bactericidal/Permeability Increasing Protein-LPS complex so isolated, so as to release the Bactericidal/ Permeability Increasing Protein from the Bactericidal/ Permeability Increasing Protein-LPS complex; and (d) isolating the Bactericidal/Permeability Increasing Protein so released. Thenon-glycosylatedBactericidal/PermeabilityIncreasing Protein produced by the method of claim 1.

Inventors: LANE JOHN C, MARRA MARIAN N, SCOTT RANDAL W, SNABLE JAMES L, LANE, JOHN, C., MARRA, MARIAN, N., SCOTT, RANDAL, W., SNABLE, JAMES, L.
Applicants: INCYTE PHARMA INC, INCYTE PHARMACEUTICALS, INC.
Application year: 1994
Main classification: C12N15
IPC codes: A01N63, A61K38, A61P31, C07K14, C12N1, C12N5, C12N15, C12P21, C12R1